The immunosuppressive activity of myeloid-derived suppressor cells in murine Paracoccidioidomycosis relies on Indoleamine 2,3-dioxygenase activity and Dectin-1 and TLRs signaling

Paracoccidioidomycosis (PCM) is a systemic mycosis with a high incidence in Latin America. Prior studies have demonstrated the significance of the enzyme Indoleamine 2,3-dioxygenase (IDO-1) in the immune regulation of PCM as well as the vital role of myeloid-derived suppressor cells (MDSCs) in moderating PCM severity. Additionally, Dectin-1 and Toll-Like Receptors (TLRs) signaling in cancer, infection, and autoimmune diseases have been shown to impact MDSC-IDO-1+ activity. To expand our understanding of MDSCs and the role of IDO-1 and pattern recognition receptors (PRRs) signaling in PCM, we generated MDSCs in vitro and administered an IDO-1 inhibitor before challenging the cells with Paracoccidioides brasiliensis yeasts. By co-culturing MDSCs with lymphocytes, we assessed T-cell proliferation to examine the influence of IDO-1 on MDSC activity. Moreover, we utilized specific antibodies and MDSCs from Dectin-1, TLR4, and TLR2 knockout mice to evaluate the effect of these PRRs on IDO-1 production by MDSCs. We confirmed the importance of these in vitro findings by assessing MDSC-IDO-1+ in the lungs of mice following the fungal infection. Taken together, our data show that IDO-1 expression by MDSCs is crucial for the control of T-cell proliferation, and the production of this enzyme is partially dependent on Dectin-1, TLR2, and TLR4 signaling during murine PCM.

www.nature.com/scientificreports/ pathologies [12][13][14] . Although few studies have addressed the involvement of MDSCs in fungal infections 15,16 , the role of MDSCs in PCM has been recently investigated by our group. Preite et al. 17 showed that MDSCs are recruited to the lungs of P. brasiliensis-infected mice and express the immunosuppressive enzyme Indoleamine 2,3 Dioxigenase-1 (IDO-1), which contributes to impaired Th1 and Th17 responses in the disease, resulting in a severe pulmonary PCM that could be reversed by MDSC depletion in mice 17 . The expression of IDO-1 is a major mechanism of immunosuppression in a variety of cancer types 18 , and the infiltration of MDSCs has been associated with this enzyme activity 19 . Furthermore, in different pathological contexts the expression of IDO-1 was already shown to be induced by pattern recognition receptors (PRRs), such as Dectin-1 20 , Toll-like receptor 2 (TLR2) 21 , and TLR4 22 . Moreover, these PPRs are involved in the activity or recruitment of MDSCs in several infectious diseases 15,23,24 . For instance, the induction of MDSCs after Aspergillus fumigatus and Candida albicans infections has been reported to be dependent on Dectin-1/CARD9 signaling, resulting in ROS generation and caspase-8 activity, along with IL-1β production 15 . The induction of inflammation via PRRs by innate immune cells requires careful control to avoid damage to host tissues. This regulation depends on intracellular receptors, membrane-bound suppressors, intracellular negative regulators, the degradation of TLRs, apoptosis induced by TLRs, and also the activity of MDSCs 23 . Intriguingly, MDSCs can be induced by TLR agonists, such as lipopolysaccharide (LPS), or whole pathogens 23 . In general, TLR2 and TLR4 cognates derived from microorganisms are capable of inducing monocytic MDSCs, as demonstrated in previous hepatitis C virus (HCV) and Staphylococcus aureus infection studies 24,25 . Specifically, TLR2 agonists, such as those present in S. aureus, can promote the differentiation of monocytes into MDSCs, leading to their accumulation in skin lesions 26,27 . In PCM, the involvement of Dectin-1, TLR2, and TLR4 has already been extensively studied [28][29][30][31][32][33][34][35][36][37][38][39][40] . However, their involvement in MDSCs recruitment and activity during pulmonary PCM has yet to be investigated. Thus, we aimed to evaluate the role of IDO-1 in MDSCs activity and the influence of Dectin-1, TLR2, and TLR4 on the production and activity of this immunosuppressive enzyme by MDSCs in a pulmonary model of P. brasiliensis infection in mice.

Methods
Mice. The experiments were performed in accordance with Brazilian Federal Law 11,794, as well as in accordance with the ARRIVE guidelines. The study was approved by the Ethics Committee on Animal Experiments of UNIFESP (Protocol Nº 2135170220). Eight-to 12-week-old male C57BL6/J WT, TLR2KO, and TLR4KO mice were bred as specific pathogen-free mice at the Center for the Development of Experimental Models for Biology and Medicine and were kept in the facility of the Institute of Science and Technology of UNIFESP. Also, eight-to 12-week-old male C57BL/6 Dectin-1KO, IDO-1KO, and WT mice were obtained from the specific pathogenfree isogenic breeding unit of the Department of Immunology, Institute of Biomedical Sciences, University of São Paulo.

Fungus and infection. The virulent P. brasiliensis 18 (Pb18 isolate) yeast cells were cultured weekly in Fava
Netto culture medium at 37 °C and used on days 7-8 of culture. The viability of yeasts, which was consistently higher than 95%, was determined using Janus Green B vital dye (Merck). Mice were anesthetized and subjected to intratracheal (it.) infection as previously described 41 . Briefly, after intraperitoneal (ip) injection of ketamine (90 mg/kg) and xylazine (10 mg/kg), animals were infected with 1 × 10 6 yeast cells in 50 μL of phosphate-buffered saline.
In vitro generation of MDSCs. Bone-marrow-derived MDSCs (BM-MDSCs) were generated from naïve C57BL/6 WT, IDO-1KO, Dectin-1KO, TLR2KO, and TLR4KO mice as previously described 42 . All animals were euthanized with intraperitoneal (ip) injection of ketamine (270 mg/kg) and xylazine (30 mg/kg). BM cells were flushed out from the femurs and tibias of mice using a 1 mL syringe and Dulbecco's Modified Eagle Medium (DMEM, Sigma-Aldrich) supplemented with 3% fetal bovine serum. Red blood cells (RBC) were lysed in RBC lysis buffer (BioLegend) for 4 min. Cells were then washed with DMEM, and 7 × 10 5 white blood cells per mL were cultured in cell culture bottles with DMEM supplemented with 10% FBS, recombinant murine IL-6, and granulocyte-monocyte colony-stimulating factor (GM-CSF), both at 10 ng/mL (Biolegend), and cultured for three days at 37 °C in a 5% CO 2 chamber. BM-MDSCs were separated from other myeloid cell populations using the MDSC-isolation kit (Miltenyi), following the manufacturer's instructions.

Immunosuppressive activity of BM-MDSCs on T-lymphocytes.
We generated single-cell suspensions from the spleens of naïve C57BL/6 WT mice. After RBC lysis, T-cell populations were separated using the Pan T-Cell Isolation Kit (Miltenyi). To evaluate the proliferation of T-lymphocytes, cells were stained with carboxyfluorescein succinimidyl ester (CellTrace™ CFSE Cell Proliferation Kit, Invitrogen) according to the manufacturer's instructions. Subsequently, 1 × 10 6 T-lymphocytes were activated with 1 µg/mL anti-CD3 and anti-CD28 monoclonal antibodies (BioLegend) and co-cultured at 37 °C and 5% CO 2 for four days in the presence or absence of 1 × 10 5 BM-MDSCs per well in a 96-well U-bottom plate. BM-MDSCs were previously challenged or not with 4 × 10 3 P. brasiliensis yeasts. The proliferation of T-cells was defined according to the CFSE dilution and assessed by flow cytometry as outlined in the Suppl. Fig. 3 17,43 .
Lung-infiltrating MDSCs in P. brasiliensis-infected mice. Lung tissues were collected from C57BL/6 WT, IDO-1KO, Dectin-1KO, TLR2KO, and TLR4KO P. brasiliensis-infected mice after 72 h, two weeks, and eight weeks of infection, respectively. The tissues were enzymatically digested in RPMI medium with 10% FBS containing 2 mg/mL of collagenase (Sigma-Aldrich) for 40 min at 37 °C and 120 rpm in a shaker incubator. Lung leukocyte suspensions were obtained as previously described 32 and subjected to cell staining as described above.

Treatment of MDSCs with an IDO-1 inhibitor (1-MT) decreased their suppressive activity on T-cells.
The treatment of MDSCs with 1MT reduced their suppressive capacity, particularly on activated CD4 + T-cells, as evidenced by high CD4 + T-cell frequencies in co-cultures with 1MT-treated MDSCs compared to untreated MDSCs ( Fig. 1A and B). This suggests that IDO-1 produced by MDSCs is involved in lymphocytes immunosuppression. The addition of MDSCs to the culture impaired the proliferation of CD8 + T-lymphocytes. Slight differences were also observed in the frequency of total CD8 + T populations as well as in activated CD8 + T-lymphocytes when MDSCs were treated or not with 1MT ( Fig. 1C and D). A higher proliferation index of the CD4 + T-lymphocytes was observed in co-culture with 1MT-treated compared with untreated controls, reflecting their elevated frequencies (Fig. 1E). Notably, the impaired suppression of CD8 + T-lymphocytes was only observed after the in vitro challenge of MDSCs with P. brasiliensis (Fig. 1F).

IDO-1 absence reduced the ability of MDSCs to suppress T-cell proliferation. IDO-1-inhibited-
MDSCs were generated in vitro from both WT and IDO-1KO mice and co-cultured with activated T-cells. Our findings indicate that the frequencies of CD4 + T and CD4 + CD25 + T-lymphocytes were higher after co-culture with IDO-1-deficient MDSCs in the presence of P. brasiliensis than with WT-MDSCs ( Fig. 2A and B). However, the absence of IDO-1 in MDSCs did not affect the frequency of CD8 + T-cells (Fig. 2C), and no differences were observed in CD8 + T-activated cells (Fig. 2D). The analysis of T-cell proliferation revealed higher proliferation indices of CD4 + T-cells co-cultured with IDO-1-deficient MDSCs previously challenged with P. brasiliensis yeasts than those of T-cells co-cultured with WT-MDSCs, indicating an impaired suppressive capacity of IDO-1-deficient MDSCs ( Fig. 2E and F). Collectively, our data demonstrate the contribution of IDO-1 to the adequate suppression of T-cell proliferation by MDSCs. Given that IDO-1 inhibition has previously been associated with decreased infiltration and activity of MDSCs in human melanoma tumors 19 , we investigated the role of IDO-1 in MDSC recruitment to the lungs of P. brasiliensis-infected WT and IDO-1KO mice. Our results showed that IDO-1 absence did not affect the frequency and number of lung-infiltrating MDSCs ( Fig. 3A and B).  Control T-cells without contact with MDSCs. Differences between groups were analyzed by analysis of variance (ANOVA) followed by the Tukey test. Results were considered significant at *p < 0.05; **p > 0.01; ***p < 0.001, and ****p < 0.0001. were cultured without contact with MDSCs. The data represent three independent experiments (N = 5 wells/ group). Differences between treatments were analyzed by analysis of variance (ANOVA) followed by the Tukey test. Results were considered significant at *p < 0.05; **p > 0.01; ***p < 0.001, and ****p < 0.0001. www.nature.com/scientificreports/ unlike M-MDSCs, no differences in the frequency and number of IDO-1 + PMN-MDSCs were observed between the two evaluated groups ( Fig. 5D and E).

Anti-TLR2 treatment or TLR2 absence reduced IDO-1 expression by BM-MDSCs in vitro.
In addition to Dectin-1, TLR2, another PPR, has been shown to affect IDO-1 production. For instance, a study has shown that fractions from Mycobacterium leprae modulated IDO-1 production by monocyte-derived DCs in a TLR2-dependent manner 21 . Considering this and prior research indicating a role for TLR2 in the activity of MDSCs 26,27 , we aimed to investigate the impact of TLR2 signaling on IDO-1 expression by MDSCs challenged with P. brasiliensis yeasts in vitro. Anti-TLR2 treatment resulted in reduced IDO-1 production by M-MDSCs after P. brasiliensis challenge compared to untreated cells. No differences were observed between anti-TLR2 treated MDSCs and controls in the absence of the fungus. Regarding PMN-MDSCs, there was an increase in the frequency of PMN-MDSC-IDO-1 + in untreated cells challenged with the fungus. However, when the MDSCs were treated with anti-TLR2 followed by a fungal challenge, the PMN-MDSCs failed to increase IDO-1 production (Fig. 6A). Our results suggest a significant role for TLR2 signaling in regulating IDO-1 production by MDSCs during the P. brasiliensis-yeast challenge. Furthermore, M-MDSCs derived from TLR2KO animals showed impaired production of IDO-1 after challenge with P. brasiliensis yeasts, compared to MDSCs from WT mice, but no changes in IDO-1 production by PMN-MDSCs in TLR2KO cells were observed (Fig. 6B).

TLR2 absence affected the expression of IDO-1 by lung-infiltrating M-and PMN-MDSCs after P. brasiliensis infection. TLR2 absence did not alter the frequency or the number of lung-infiltrating
MDSCs after infection when compared to WT controls ( Fig. 6C and D). However, when the expression of IDO-1 was analyzed, a reduced frequency of M-MDSCs expressing IDO-1 was detected 72 h and two weeks post-P. brasiliensis infection. Also, a decreased number of IDO-1-producing M-MDSCs was observed after two weeks www.nature.com/scientificreports/ of infection (Fig. 7A). TLR2KO mice showed a reduced frequency of lung-infiltrating PMN-MDSCs expressing IDO-1 at two-and eight-weeks post infection, along with a reduced number of these cells after 2 weeks of infection, compared to wild-type controls (Fig. 7B).
Diminished IDO-1 expression by BM-MDSCs in response to in vitro anti-TLR4 treatment and TLR4 absence. TLR4, an innate immune receptor, has an impact on both IDO-1 expression 22 , and MDSC activity 25 . In this context, we investigated the contribution of TLR4 to IDO-1 expression by MDSCs in response to a P. brasiliensis-yeast challenge in vitro. Anti-TLR4 treatment did not affect IDO-1 production by M-MDSCs. However, in PMN-MDSCs, IDO-1 expression increased after fungal challenge, but this increase was absent in cells that received anti-TLR4 treatment (Fig. 8A). These data suggest that TLR4 signaling is involved in IDO-1 www.nature.com/scientificreports/ expression by PMN-MDSCs. In further assays, M-MDSCs from TLR4KO mice produced less IDO-1 after challenge with P. brasiliensis yeasts, while no such reduction was observed in MDSCs from WT mice. In addition, PMN-MDSCs deficient in TLR4 also displayed impaired IDO-1 production (Fig. 8B), consistent with the results obtained with TLR4-blocked MDSC.

TLR4 deficiency reduces IDO-1 expression by lung-infiltrating MDSCs in response to P. brasiliensis infection. TLR4 deficiency did not alter the frequency or number of lung-infiltrating MDSCs post
infection, compared to WT controls ( Fig. 8C and D). However, we did observe a decreased frequency of IDO-1 + M-MDSCs at 72 h post infection in TLR4-deficient mice compared to WT controls (Fig. 9A). Additionally, we detected a reduced frequency of PMN-MDSCs expressing IDO-1 in TLR4KO mice at eight weeks post infection (Fig. 9B).

Discussion
Despite a lack of studies investigating the role of IDO-1 production by MDSCs in microbial infections, recent research has shown that other cell populations have a remarkable role in this regard. For instance, we have previously demonstrated the immunosuppressive activity of plasmacytoid dendritic cells (pDCs) via an IDO-1-dependent mechanism. The depletion of pDC-IDO-1 + resulted in the most severe manifestation of PCM 45 .
Here, we showed that the inhibition of IDO-1 by 1MT in MDSCs co-cultured with lymphocytes resulted in a higher rate of T-cell proliferation compared to untreated MDSCs. These findings suggest that, despite the activity of other immunosuppressive mechanisms in MDSCs during P. brasiliensis infection 17 , IDO-1 contributes to the suppressive activity of MDSCs. Interestingly, IDO-1 expression did not enhance MDSC differentiation or influx towards the lungs of P. brasiliensis-infected mice. Nonetheless, our data support the previously observed reduced suppressive activity of MDSCs after IDO-1 inhibition in cancer 19,[46][47][48] .
In autoimmune and infectious diseases, it has been demonstrated that PRRs, such as Dectin-1 and TLRs, can influence IDO-1-dependent or IDO-1-independent mechanisms of MDSC 15,23,24,49 . Rieber et al. 15 showed that the activation of the Dectin-1/CARD9 pathway can stimulate the suppressive activity of PMN-MDSCs in the immune response against the pathogenic fungi A. fumigatus and C. albicans 15,16 . Here, we demonstrated that Dectin-1 is an important receptor for the adequate expression of IDO-1 by MDSCs in PCM. Thus, the lower number of IDO-1-producing M-MDSCs in the lungs of Dectin-1KO animals may be attributed to the pulmonary microenvironment. Furthermore, the C-type lectin-like receptor-2d, which recognizes glucuronoxylomannan, an abundant polysaccharide component of Cryptococcus neoformans, is fundamental in the recruitment of PMN-MDSCs in mice and patients with cryptococcosis 50 . These studies highlight the promising role of C-type lectin receptors expressed by MDSCs as immunotherapeutic targets in the treatment of fungal diseases.
The role of TLR signaling in MDSCs has been studied in several infectious diseases 24,26,27,49,51,52 . However, the role of TLR2 signaling in IDO-1 production by MDSCs, previously demonstrated in other cell populations, has not been thoroughly investigated. In Mycobacterium leprae infection, a decrease in IDO-1 activity was observed after TLR2 neutralization by monocyte-derived DCs 21 . Our results showed decreased IDO-1 production in TLR2inhibited MDSCs after P. brasiliensis challenge. The reduction of IDO-1 expression by MDSCs obtained from P. brasiliensis-infected TLR2KO mice is consistent with that obtained with BM-MDSCs and TLR2 neutralization. Although our data demonstrate a correlation between TLR2 signaling and IDO-1 expression, TLR2 deficiency did not influence the recruitment of lung-infiltrating MDSCs. Interestingly, TLR2 deficiency resulted in elevated IL-6 levels in the lungs of P. brasiliensis-infected mice 29 , a cytokine required for MDSC expansion and influx Differences between treatments were analyzed by analysis of variance (ANOVA) followed by the Tukey test. The data presented in this figure were obtained from three independent in vivo experiments with 3-5 mice per group. For comparisons between two groups, the mean ± SEM was obtained and analyzed by the unpaired Student's t-test. Differences were considered significant at *p < 0.05; **p > 0.01 ***p < 0.001, and ****p < 0.0001.  44,53 . Such elevated IL-6 levels could compensate for a possible negative impact on MDSC recruitment due to reduced IDO-1 expression by these cells. Notably, our data are consistent with a study with Porphyromonas gingivalis, the etiologic agent of periodontitis, where MDSC induction was not affected by TLR2 deficiency in mice 54 . Elevated levels of IDO-1 can activate mature Treg cells via activation of the protein kinase general control nonderepressing-2 pathway of protein synthesis inhibition. Besides, IDO-1 produced by pDCs activates Treg cells and can further convert naïve T-cells into new Treg cell subsets 45,55 . Therefore, considering that MDSCs are an important source of IDO-1 17 , the reduced expression of this tolerogenic enzyme by lung-infiltrating Mand PMN-MDSCs in TLR2-defective mice could account for the lower expansion of Treg cells as previously described 29 .

Scientific Reports
In line with our observations on TLR2, we found no differences in the recruitment of lung-infiltrating MDSCs between WT and TLR4KO P. brasiliensis-infected mice. These findings coincide with a previous study of P. Figure 8. The role of TLR4 signaling in MDSCs recruitment and in IDO-1 production by MDSCs. To evaluate whether TLR4 signaling plays a role in IDO-1 production by MDSCs, MDSCs were generated in vitro from C57BL/6 WT mice and cultured in 96-well plates (2 × 10 5 per well). The cells were subsequently challenged with P. brasiliensis yeasts at a 1:25 ratio (yeast: MDSCs) overnight or not. MDSCs were treated or not with 10 µg/ mL of anti-TLR4 or IgG2b for 2 h before the fungal challenge (A). In other experiments, MDSCs were generated from WT and TLR4 KO mice, following the aforementioned fungal challenge (B). (C and D) WT and TLR4 KO mice were infected intratracheally with 1 × 10 6 P. brasiliensis yeasts. Lungs were collected 72 h, two weeks, and eight weeks post infection. Specific antibodies conjugated to fluorochromes, as shown in Suppl. Fig. 1, were used to characterize MDSC subpopulations. The frequency and total cell count of lung-infiltrating M-MDSCs (C) and PMN-MDSCs (D) 72 h, two weeks, and eight weeks post infection were determined by comparing WT with TLR4 KO. The data represent three independent experiments with 3-6 mice each. For comparisons between two groups, the mean ± SEM was obtained and analyzed by the unpaired Student's t-test. D.  25,54 , yet notable discoveries have arisen in the realm of cancer and inflammatory pathologies. For instance, chronic inflammation has been associated with the recruitment and activity of MDSCs, which can enhance the malignancy of the tumor cells. Interestingly, through co-culture experiments using MDSCs and macrophages derived from WT and TLR4KO BALB/c mice, it was demonstrated that cross-talk between inflammation-induced MDSCs and LPS-activated macrophages was TLR4-dependent. As MDSCs from TLR4KO mice failed to produce IL-10 and were less effective in reducing macrophage production of IL-12, the TLR4 pathway in MDSCs was considered a promising therapeutic target for promoting anti-tumor immune responses 56 .

Scientific
To the best of our knowledge, we present the first study evaluating the role of TLR4 signaling and IDO-1 production by MDSCs in fungal infection, notwithstanding intriguing outcomes documented in other cell populations. Notably, TLR4 engagement in LPS-primed DCs under endotoxin tolerance conditions elevated levels of IDO isoforms, IL-10, and programmed death ligands 1 and 2 expression, which were contingent upon IDO, upon investigating IDO's role in inducing DC phenotype and function 57 .
Loures et al. 30 revealed that TLR4-deficient mice displayed an upsurge in Treg cells in their lungs following P. brasiliensis infection, unlike their TLR2-deficient counterparts 29 . However, this increase was ascribed to reduced pulmonary fungal loads, leading to a decline in the influx of inflammatory cells to the site of infection 30 . Of note, the differences in the frequency of lung-infiltrating IDO-1 + MDSCs between WT and TLR4KO mice observed here were not accompanied by differences in the total cell numbers, which could indicate a weak effect of TLR4 signaling for IDO-1 production by MDSCs during P. brasiliensis infection. These findings raise the importance of further studies addressing other regulatory molecules produced by MDSCs in PCM and other infectious diseases.
It is well established that the activation of different PRRs programs DCs to induce preferential Th1, Th2, Th17, or Treg responses. In PCM, TLR2 signaling induced by P. brasiliensis infection promotes anti-inflammatory responses (IL-10, TGF-β) and Treg expansion 29 . Conversely, TLR4 signaling induces a pro-inflammatory response that favors the expansion of Th1 and Th17 cells 30 . The activation of Dectin-1 and NLRP3 induces the proliferation of Th1 and Th17 lymphocytes and inhibits the expansion of Treg cells 32 . However, it is well known that IDO-1 can be induced by both pro-and anti-inflammatory mechanisms mediated by IFN-γ and TGF-β. Our previous studies have shown that IDO-1 expression by pulmonary DCs can be mediated by IFN-γ, which uses the canonical pathway (p65, 50) of the transcription factor NFκB, as well as by TGF-β, which uses the non-canonical pathway (p52, RelB) of NFκB activation 45 . Thus, it is understandable how blocked PRRs with antagonistic effects, such as those mediated by TLR2, TLR4, and Dectin-1 signaling, lead to a common effect: reduced IDO-1 expression by MDSCs. Figure 9. To verify whether TLR4 signaling plays a role in IDO-1 production by lung infiltrating MDSCs, WT and TLR4 KO mice were infected intratracheally with 1 × 10 6 P. brasiliensis yeasts. Lungs were collected 72 h, two weeks, and eight weeks post infection. The expression of IDO-1 by M-MDSCs (A) and PMN-MDSCs (B) in the lungs was assessed. The data presented in this figure were obtained from three independent in vivo experiments with 3-6 mice per group. Differences between treatments were analyzed by analysis of variance (ANOVA) followed by the Tukey test. For comparisons between two groups, the mean ± SEM was obtained and analyzed by the unpaired Student's t-test. Differences were considered significant at **, p < 0.001, and ****p < 0.0001.

Conclusion
Taken together, our findings demonstrate that IDO-1 expression by MDSCs is an important mechanism for controlling T-cell proliferation and is partially dependent on Dectin-1, TLR2, and TLR4 signaling in the pulmonary PCM. Understanding the regulators and signaling pathways involved in the differentiation and activation of MDSCs is essential for developing new immunotherapeutic strategies targeting MDSCs, which has become a promising tool since our previous study has demonstrated 17 that these cell populations are a potential target for PCM control.

Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.